Bottom Line:
These mitotic problems are not caused by overall defects in mitotic NPC disassembly-reassembly or general nuclear import.However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis.Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.

Figure 1: Identification of a novel Nup via affinity purification of Nup2. (A) Western blot of A. nidulans total cell extracts from wild-type strain R153 using Nup2 antibody pretreated with or without the Nup2 antigen (Ag) as indicated. The antibody recognizes Nup2 during both G2 and mitosis (M). (B) Cells of strain R153 were fixed and labeled with the Nup2 antibody and DAPI stained to visualize DNA. Native Nup2 is at NPCs during interphase but localized near DNA during mitosis. Scale bar, ∼5 μm. (C) Coomassie-stained SDS–PAGE gel showing Nup2 copurified with a novel nucleoporin termed NupA identified by mass spectroscopy (from strain SO926). The first two samples depict affinity purifications performed for G2 and mitotic samples. The third purified mitotic sample was treated with λ phosphatase before electrophoresis. Sequence coverage obtained from mass spectroscopy: G2 sample: 72.9% for Nup2, 34.2% for NupA, 37.8% for KapB, and 36.2% for KapA; mitotic sample: 73% for Nup2, 36.7% for NupA, 34.1% for KapB, and 32.4% for KapA. (D) Cartoon depicting genomic structure of nupA from the indicated species. All contain a large intron between exons 1 and 2.

Mentions:
Endogenous Nup2 tagged with green fluorescent protein (GFP) is functional and translocates from NPCs to the chromatin region during mitotic entry and relocates back to NPCs during mitotic exit (Osmani et al., 2006a). To determine whether Nup2 is located like Nup2-GFP, an antibody was generated and used for immunofluorescence microscopy. Similar to the GFP-tagged version, untagged Nup2 was found to locate to NPCs during interphase and the chromatin region during mitosis (Figure 1, A and B), indicating Nup2-GFP reflects the normal cell cycle– regulated locations of Nup2.

Figure 1: Identification of a novel Nup via affinity purification of Nup2. (A) Western blot of A. nidulans total cell extracts from wild-type strain R153 using Nup2 antibody pretreated with or without the Nup2 antigen (Ag) as indicated. The antibody recognizes Nup2 during both G2 and mitosis (M). (B) Cells of strain R153 were fixed and labeled with the Nup2 antibody and DAPI stained to visualize DNA. Native Nup2 is at NPCs during interphase but localized near DNA during mitosis. Scale bar, ∼5 μm. (C) Coomassie-stained SDS–PAGE gel showing Nup2 copurified with a novel nucleoporin termed NupA identified by mass spectroscopy (from strain SO926). The first two samples depict affinity purifications performed for G2 and mitotic samples. The third purified mitotic sample was treated with λ phosphatase before electrophoresis. Sequence coverage obtained from mass spectroscopy: G2 sample: 72.9% for Nup2, 34.2% for NupA, 37.8% for KapB, and 36.2% for KapA; mitotic sample: 73% for Nup2, 36.7% for NupA, 34.1% for KapB, and 32.4% for KapA. (D) Cartoon depicting genomic structure of nupA from the indicated species. All contain a large intron between exons 1 and 2.

Mentions:
Endogenous Nup2 tagged with green fluorescent protein (GFP) is functional and translocates from NPCs to the chromatin region during mitotic entry and relocates back to NPCs during mitotic exit (Osmani et al., 2006a). To determine whether Nup2 is located like Nup2-GFP, an antibody was generated and used for immunofluorescence microscopy. Similar to the GFP-tagged version, untagged Nup2 was found to locate to NPCs during interphase and the chromatin region during mitosis (Figure 1, A and B), indicating Nup2-GFP reflects the normal cell cycle– regulated locations of Nup2.

Bottom Line:
These mitotic problems are not caused by overall defects in mitotic NPC disassembly-reassembly or general nuclear import.However, without Nup2 or NupA, although the SAC protein Mad1 locates to its mitotic locations, it fails to locate to NPCs normally in G1 after mitosis.Collectively the study provides new insight into the roles of Nup2 and NupA during mitosis and in a surveillance mechanism that regulates nucleokinesis when mitotic defects occur after SAC fulfillment.